Production of chimeric antibodies by homologous recombination

ABSTRACT

A process for producing chimeric antibodies using novel recombinant DNA vectors and homologous recombination in vivo is described. The recombinant DNA constructs of the invention can be used to transfect antibody producing cells so that targeted homologous recombination occurs in the transfected cells leading to gene modification and the production of chimeric antibody molecules by the transfected cells.

The present application is a continuation-in-part of copendingapplication Ser. No. 07/242,873, filed Sep. 14, 1988, and of applicationSer. No. 113,800, filed Oct. 27, 1987, now abandoned, both of which areincorporated by reference herein in its entirety.

TABLE OF CONTENTS

1. Introduction

2. Background of the Invention

2.1. Chimeric Antibodies

2.2 Homologous Recombination

3. Summary of the Invention

3.1 Definitions

4. Description of the Figures

5. Detailed Description of the Invention

5.1. Target Vectors

5.1.1. Target Sequence

5.1.2. Replacement Gene

5.1.3. Selectable Marker and Other Elements

5.2. Transfection of Antibody-Producing Cell Lines With Target Vectors

5.3. Screening and Selection of Recombinants

6. Example: Replacement Of The Constant Region Of A MurineImmunoglobulin Heavy Chain With The Constant Region of Human Gamma 1Immunoglobulin

6.1. Materials and Methods

6.1.1. Transfection

6.1.2. Screening Transfectants for Successful Recombinants

6.1.3. Southern and Western Blots

6.2. Construction of Target Plasmids Encoding Human ImmunoglobulinConstant Region

6.3. Transfection and Homologous Recombination

6.3.1. Co-Transfection of Murine Myeloma Cells

6.3.2. Identification of Cells Secreting Chimeric Immunoglobulins

7. Example: Production of Chimeric Immunoglobulin Heavy Chain WithSpecificity To L20 Human Tumor Associated Antigen

7.1. Construction of Target Vector and Transfection of Hybridoma L20

7.2. Characterization of Expressed Chimeric L20 Heavy Chain

7.2.1. Western Blot Analysis

7.2.2. Flow Cytometric Analysis

7.2.3. Southern Blot Analysis

7.2.4. ADCC Analysis

7.3. Chimeric L20 Heavy Chain Produced Using A Different Target Vector

8. Construction of Chimeric G28.1 Heavy Chains

9. Construction of Chimeric L6 Heavy Chains

10. Vectors Useful For Production Of Chimeric Antibodies

11. Deposit Of Microorganisms

1. INTRODUCTION

The present invention relates to a process for producing chimericantibodies using novel recombinant DNA vectors and homologousrecombination in situ. The recombinant DNA constructs of the inventioncan be used to transfect antibody producing cells so that targetedhomologous recombination occurs in the transfected cells leading to theproduction of chimeric antibody molecules by the transfected cells.

The invention is demonstrated by way of examples described in which theconstant regions of a murine immunoglobulin heavy chain were replaced byhuman IgG1 constant regions and chimeric heavy chains were produced bythe murine cell line.

2. BACKGROUND OF THE INVENTION 2.1. Chimeric Antibodies

Since the development of the cell fusion technique for the production ofmonoclonal antibodies (Kohler and Milstein, 1975, Nature (London)256:495) a vast number of monoclonal antibodies, many of which defineheretofore unknown antigens, have been produced by a number ofresearchers. Unfortunately, most of the monoclonal antibodies made todate are produced in a murine system and, therefore, have limitedutility as human therapeutic agents unless modified in some way so thatthe murine monoclonal antibodies are not "recognized" as foreignepitopes and "neutralized" by the human immune system. A number ofresearchers, therefore, are attempting to develop human monoclonalantibodies, which are "recognized" less well as foreign epitopes and mayovercome the problems associated with the use of monoclonal antibodiesin humans. Obviously, the hybridoma technique developed by Kohler andMilstein (supra) which involves sacrificing the immunized mice and usingtheir spleens as a source of lymphocytes for subsequent fusion toimmortalize antibody producing cell lines cannot be practiced in humans.Therefore, a number of researchers have directed their attention torecent advances in the field of molecular biology that allow for theintroduction of DNA into mammalian cells to obtain expression ofimmunoglobulin genes (Oi et al., 1983 Proc. Natl. Acad. Sci. USA 80:825;Potter et al., 1984, Proc. Natl. Acad. Sci. USA 81:7161), and have usedthese techniques to produce chimeric antibodies (Morrison et al., 1984,Proc. Natl. Acad. Sci. USA 81:6581; Sahagan et al. 1986, J. Immunol.137:1066; Sun et al., 1987, Proc. Natl. Acad. Sci. 84:214).

Chimeric antibodies are immunoglobulin molecules comprising a human andnon-human portion. More specifically, the antigen combining region (orvariable region) of a chimeric antibody is derived from a non-humansource (e.g., murine) and the constant region of the chimeric antibody(which confers biological effector function to the immunoglobulin) isderived from a human source. The chimeric antibody should have theantigen binding specificity of the non-human antibody molecule and theeffector function conferred by the human antibody molecule.

In general, the procedures used to produce these chimeric antibodiesconsist of the following steps (the order of some steps may beinterchanged):

(a) identifying and cloning the correct gene segment encoding theantigen binding portion of the antibody molecule; this gene segment(known as the VDJ, variable, diversity and joining regions for heavychains or VJ, variable, joining regions for light chains (or simply asthe V or Variable region) may be in either the cDNA or genomic form;

(b) cloning the gene segments encoding the constant region or desiredpart thereof;

(c) ligating the variable region with the constant region so that thecomplete chimeric antibody is encoded in a transcribable andtranslatable form;

(d) ligating this construct into a vector containing a selectable markerand gene control regions such as promoters, enhancers and poly(A)addition signals;

(e) amplifying this construct in bacteria;

(f) introducing the DNA into eukaryotic cells (transfection) most oftenmammalian lymphocytes;

(g) selecting for cells expressing the selectable marker;

(h) screening for cells expressing the desired chimeric antibody; and

(i) testing the antibody for appropriate binding specificity andeffector functions.

Antibodies of several distinct antigen binding specificities have beenmanipulated by these protocols to produce chimeric proteins (e.g.,anti-TNP: Boulianne et al., 1984, Nature Vol. 312 pg. 643; andanti-tumor antigens: Sahagan et al., 1986, J. Immunol. Vol. 137:1066).Likewise several different effector functions have been achieved bylinking new sequences to those encoding the antigen binding region. Someof these include enzymes (Neuberger et al., 1984, Nature 312:604),immunoglobulin constant regions from another species and constantregions of another immunoglobulin chain (Sharon et al., 1984, Nature309:364; Tan et al., 1985, J. Immunol. Vol. 135:3565-3567).

2.2. Homologous Recombination

Another recent advance in the field of molecular biology is thediscovery that cultured mammalian cells will integrate exogenous plasmidDNA into chromosomal DNA at the chromosome location which containssequences homologous to the plasmid sequences. This event is referred toas homologous recombination (Folger, et al. 1982, Mol. Cell. Biol. 2,1372-1387; Folger, et al., 1984, Symp. Quant. Biol. 49, 123-138;Kucherlapati, et al., 1984, Proc. Natl. Acad. Sci. USA 81, 3153-3157;Lin, et al., 1985, Proc. Natl. Acad. Sci. USA 82, 1391-1395; Robert deSaint Vincent, et al., 1983, Proc. Natl. Acad. Sci. USA 80,2002-2006;Shaul, et al., 1985, Proc. Natl. Acad. Sci. USA 82, 3781-3784).Mammalian cells also contain the enzymatic machinery to integrateplasmid DNA at random chromosomal sites, referred to as nonhomologousrecombinations. The frequency of homologous recombination has beenreported to be as high as between 1/100 to 1/1000 of the recombinationalevents, while the majority of recombinations result from nonhomologousinteractions (Thomas et al., 1986, Cell 44:419-428; Smithies et al.,1985, Nature 317:230-234; Shaul, et al., 1985, Proc. Natl. Acad. Sci.USA 82, 3781-3784; Smith, et al., 1984, Symp. Quant. Biol. 49, 171-181;Subramani, et al., 1983 Mol. Cell. Biol. 3, 1040-1052). The existence ofthe cell machinery for homologous recombination makes it possible tomodify endogenous genes in situ. In some instances, conditions have beenfound where the chromosomal sequence can be modified by introducing intothe cell a plasmid DNA which contains a segment of DNA homologous to thetarget locus and a segment of new sequences with the desiredmodification (Thomas et al., 1986, Cell 44:419-428; Smithies et al.,1985, Nature 317:230-234; Smith, et al., 1984, Symp. Quant. Biol. 49,171-181). Homologous recombination between the mammalian cellchromosomal DNA and the exogenous plasmid DNA can result in theintegration of the plasmid or in the replacement of some of thechromosomal sequences with homologous plasmid sequences. The process ofreplacing homologous DNA sequences is referred to as gene conversion.Both the integration and the conversion events can result in positioningthe desired new sequence at the endogenous target locus.

However, the process of homologous recombination has mostly been studiedusing genes which offer dominant selection such as NEO and HPRT and onlyfor a very few cell types (Song et al., 1987, Proc. Natl. Acad. Sci. USA84:6820-6824; Rubinitz and Subramani, 1986, Mol. Cell Biol. 6:1608-1614;and Liskay, 1983, Cell 35:157-164). It has not been determined whetheror not lymphocytes or myeloma cells are capable of mediating such eventsor whether immunoglobulin genes could be usefully targeted orreconstructed by such a process.

3. SUMMARY OF THE INVENTION

The present invention is directed to a process for modifying antibodymolecules and for creating and producing chimeric antibody molecules inwhich the antigen combining region is linked (a) to an immunoglobulinconstant region or some portion thereof, that confers a desiredcharacteristic such as effector function, class (e.g., IgG, IgA, IgM,IgD or IgE) origin (e.g., human or other species); or (b) to anothertype of molecule conferring some other function to the chimeric antibodymolecule (e.g., an enzyme, toxin, a biologically active peptide, growthfactor inhibitor, or linker peptide to facilitate conjugation to a drug,toxin, or other molecule, etc.).

The invention uses novel recombinant DNA vectors to engineer targetedgene modification accomplished via homologous recombination, in either(a) cell lines that produce antibodies having desired antigenspecificities, so that the antigen combining site of an antibodymolecule remains unchanged, but the constant region of the antibodymolecule, or a portion thereof, is replaced or altered; or (b) celllines that produce antibodies of desired classes which may demonstratedesired effector functions, so that the constant region of an antibodymolecule remains unchanged, but the variable region of the antibodymolecule or a portion thereof, is replaced or altered.

According to one embodiment of the invention, a novel recombinant DNAvector is used to transfect a cell line that produces an antibody havinga desired antigen specificity. The novel recombinant DNA vector containsa "replacement gene" to replace all or a portion of the gene encodingthe immunoglobulin constant region in the cell line (e.g., a replacementgene may encode all or a portion of a constant region of a humanimmunoglobulin, a specific immunoglobulin class, or an enzyme, a toxin,a biologically active peptide, a growth factor, inhibitor, or a linkerpeptide to facilitate conjugation to a drug, toxin, or other molecule,etc.), and a "target sequence" which allows for targeted homologousrecombination with immunoglobulin sequences within the antibodyproducing cell. In an alternate embodiment of the invention, a novel DNAvector is used to transfect a cell line that produces an antibody havinga desired effector function, in which case, the replacement genecontained in the novel recombinant vector may encode all or a portion ofa region of an antibody molecule having a desired antigen specificity,and the target sequence contained in the recombinant vector allows forhomologous recombination and targeted gene modification within theantibody producing cell. In either embodiment, when only a portion ofthe variable or constant region is replaced, the resulting chimericantibody may define the same antigen and/or have the same effectorfunction yet be altered or improved so that the chimeric antibody maydemonstrate a greater antigen specificity, greater affinity bindingconstant, increased effector function, or increased secretion andproduction by the transfected antibody producing cell line, etc.Regardless of the embodiment practiced, the processes of selection forintegrated DNA (via a selectable marker), screening for chimericantibody production, and cell cloning, can be used to obtain a clone ofcells producing the chimeric antibody.

Thus, a piece of DNA which encodes a modification for a monoclonalantibody can be targeted directly to the site of the expressedimmunoglobulin gene within a B-cell or hybridoma cell line. DNAconstructs for any particular modification may be used to alter theprotein product of any monoclonal cell line or hybridoma. Such aprocedure circumvents the costly and time consuming task of cloning bothheavy and light chain variable region genes from each B-cell cloneexpressing a useful antigen specificity. In addition to circumventingthe process of cloning variable region genes, the level of expression ofchimeric antibody should be higher when the gene is at its naturalchromosomal location rather than at a random position.

3.1. Definitions

The following terms, as used herein, whether in the singular or plural,shall have the meanings indicated:

Chimeric Antibody: an antibody molecule in which (a) the constantregion, or a portion thereof, is altered, replaced or exchanged so thatthe antigen binding site (variable region) is linked to a constantregion of a different or altered class, effector function and/orspecies, or an entirely different molecule which confers new propertiesto the chimeric antibody, e.g., an enzyme, toxin, hormone, growthfactor, drug, etc.; or (b) the variable region, or a portion thereof, isaltered, replaced or exchanged with a variable region having a differentor altered antigen specificity.

Replacement Gene: a gene that encodes a product to translationallyreplace all or a portion of either the constant region or variableregion of an antibody molecule to form a chimeric antibody molecule.Replacement genes are constructed into novel recombinant DNA targetvectors of the invention which are used to transfect antibody-producingcell lines. For the modification of all or a portion of a constantregion, replacement genes of the invention may include, but are notlimited to an immunoglobulin constant region having a particulareffector function, class and/or origin (e.g., IgG, IgA, IgM, IgD, or IgEconstant regions of human immunoglobulins or any other species) or aportion of a constant region which modifies the activity or propertiesof the constant region of the immunoglobulin; as well as genes whichencode other molecules that confer some new function to a chimericantibody molecule, e.g., an enzyme, toxin, hormone, growth factor,conjugatable linker, etc. For the modification of all or a portion of avariable region, replacement genes of the invention may include, but arenot limited to immunoglobulin variable regions that encode a differentvariable region having a different antigen affinity or specificity, or aportion of a variable region which modifies the activity or propertiesof the variable region of the immunoglobulin so that the resultingchimeric antibody has a greater affinity or higher degree of specificityfor the antigen.

Target Sequence: a sequence homologous to DNA sequences that flank oroccur adjacent to the region to be converted of an immunoglobulin genesequence which may be within the gene itself or upstream or downstreamof coding sequences in the chromosome of a lymphoid cell. Targetsequences are constructed into novel recombinant DNA vectors of theinvention which are used to transfect antibody-producing cell lines.

Target sequences for heavy chain recombinations that direct replacementof or insertion within all or a portion of the constant region mayinclude but are not limited to all or a portion of the V, D, J, andswitch region (including intervening sequences called introns) andflanking sequences associated with or adjacent to the particular heavychain constant region gene expressed by the antibody producing cell lineto be transfected and may include regions located within or downstreamof the constant region (including introns). Target sequences for lightchain recombinations that direct replacement of or insertion within allor a portion of the constant region may include but are not limited tothe V and J regions, their upstream flanking sequences, and interveningsequences (introns), associated with or adjacent to the light chainconstant region gene expressed by the antibody producing cell line to betransfected and may include regions located within or downstream of theconstant region (including introns).

Target sequences for heavy chain recombinations that direct replacementof or insertion within all or a portion of the variable region mayinclude but are not limited to all or a portion of the V, D, and Jregions (including introns) and flanking sequences associated with oradjacent to the particular variable region gene expressed by theantibody producing cell line to be transfected. Target sequences forlight chain recombinations that direct replacement of or insertionwithin all or a portion of the variable region may include but are notlimited to the V and J region (including introns) and flanking sequencesassociated with or adjacent to the light chain variable region geneexpressed by the antibody producing cell line to be transfected.

Target Vector: a recombinant nucleotide vector comprising a targetsequence and a replacement gene which can be used to engineer theproduction of chimeric antibodies by lymphoid cells transfected with thetarget vector. The target vectors are used to transfect cell lines thatcontain sequence(s) homologous to the vector's target sequence and thatare capable of producing immunoglobulin molecules having (a) a desiredantigen specificity; (b) a desired constant region; or (c) anotherdesired quality such as high secretion levels, large scale cultureadaptability, etc. Such cell lines include, but are not limited to,lines derived from hybridomas which produce monoclonal antibody having adesirable specificity or effector function, as well as hybridoma cellswhich themselves have sustained mutations which prevent expression ofheavy and/or light chains of immunoglobulin.

The following abbreviations shall have the meanings shown below:

DHFR: dihydrofolate reductase

FITC: fluorescein isothiocyanate

gpt: guanosine phosphoryl transferase

HRP: horseradish peroxidase

hu: human

huCγ1: constant region exons of human gamma immunoglobulin 1

huIgG: human gamma immunoglobulin

m: mouse

mAB: monoclonal antibody

mIgG: mouse gamma immunoglobulin

4. DESCRIPTION OF THE FIGURES

FIG. 1 diagrammatically represents a generalized scheme for genereplacement via homologous recombination using the target vectors of theinvention. Variable (V), diversity (D), joining (J), switch (S) andconstant (C) regions are indicated. Panel A schematically represents thereplacement of all or a portion of the constant or variable region ofthe heavy chain genes using a target sequence homologous to any portionspanning the V, D, J, S, and C region. Panel B schematically representsthe replacement of all or a portion of the constant or variable regionof the light chain genes using a target sequence homologous to anyportion spanning the V, J, and C region. Panel C schematicallyrepresents the replacement of all or a portion of the variable orconstant regions in either light or heavy chain genes using sequencesthat flank the gene to be replaced.

FIG. 2 diagrammatically represents the construction of a target plasmidpRSV-140-neo/HuC-gamma 1/MuJ4/e. The target sequence comprises a 2.2 kbHind III fragment derived from the murine immunoglobulin heavy chain(IgH) locus which contains the fourth joining region (J4), the IgHenhancer (e) and intronic sequences 5' to the switch region. The targetsequence is positioned 5' to the replacement gene which comprises a 7.0kb Hind III-Bam HI fragment of the human IgG1 heavy chain locuscontaining the human gamma 1 constant region.

FIG. 3--Map of human IgG1 recombination vector. The vector is comprisedof a 2.2 kb Hind III fragment containing the murine heavy chain enhancerwhich has been modified to remove an Xba I site, a 3.0 kb Hind III/PvuII fragment containing the constant region exons of human gamma 1(huCγ1), the 2.0 kb Bgl II/Bam HI fragment derived from pSV2/neo whichencodes neo, a 667 bp Bal I/Sst I fragment bearing the human CMVenhancer and promoter, and a 2.3 kb Pvu II/Hind III fragment from pBR322which includes the origin of replication and the ampicillin resistancegene. The Bal I/Sst I fragment containing CMV was transferred into theSst I/Hinc III sites of pEMBL 18, removed as a Hind III/Sst I fragmentand cloned between the same sites in Pic19R, then isolated as a SmaI/Bgl II fragment to place between the Pvu II and Bgl II sites in theconstruct. Similarly, the Hind III/Pvu II fragment bearing the human cgamma 1 exons was cloned into the Hind III and Hinc II sites of Puc 9,then transferred as a Hind III/Bam HI fragment.

FIG. 4--Two additional plasmids were used for targeting the human cgamma 1 sequences. Both have the 500 bp Bgl II/Pvu II fragmentcontaining the SV40 enhancer and promoter from pSV2neo (J. Molec. Appl.Genet. I:327-341, 1982) in place of the CMV promoter and enhancer. Oneplasmid, like the plasmid of FIG. 3, has a 2.8 kb fragment encodinghuman c gamma 1, the other (shown), has the 7kb Hind III/Bam HI fragmentencoding human c gamma 1 with more sequence downstream of the constantregion exons.

FIG. 5--Vector pSV₂ gpt/Cκ for production of murine kappa light chainsby homologous recombination, containing the bacterial gpt gene.

FIG. 6--Vector pSV₂ D⁴ /C.sub.κ containing a mutated version of DHFR.

FIG. 7--Vector hγ₁ HC-D, a human gamma 1 heavy chain dissociationvector.

FIG. 8--Vector LC-D, a human C.sub.κ light chain dissociation vector.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a process for producing chimericantibodies using novel recombinant DNA vectors to direct a desiredsequence alteration to a specific location within the genome viahomologous recombination in vivo. Using the method of the invention, theprotein sequence of an antibody molecule can be modified by transfectionof a lymphoid cell line with an appropriate "target vector." In oneembodiment of the invention, a cell line that comprises immunoglobulingenes encoding a desired antigen specificity is transfected with atarget vector of the invention which comprises a recombinant DNAmolecule that encodes a "replacement gene" and a "target sequence." Thereplacement gene encodes the desired molecule which is to replace all ora portion of the constant region of the antibody molecule expressed bythe cell line. For example, the replacement gene may encode all or aportion of an immunoglobulin constant region having a particulareffector function, class and/or origin, including but not limited toIgG, IgA, IgM, IgD or IgE constant regions of human or any other desiredspecies; alternatively, the replacement gene may encode another type ofmolecule which may confer some other function to the resultant chimericantibody; e.g., an enzyme, toxin, a biologically active peptide, growthfactor, inhibitor, conjugatable peptide linker, etc. The target sequenceof the target vector is homologous to DNA sequences found in thechromosome within or adjacent to the mature gene coding sequence for theconstant region of the immunoglobulin encoded by the cell line to betransfected or an appropriate portion of the mature coding sequence forthe constant region. After transfection, homologous recombination withinthe lymphoid cell line will occur; some of these recombination eventswill lead to the replacement of all or a portion of the constant regionof the immunoglobulin gene with the replacement gene, and, therefore,the expression of chimeric antibody molecules by the transfected cells.

In an alternate embodiment of the invention, a lymphoid cell line whichcomprises an immunoglobulin gene encoding a desired constant region istransfected with a target vector containing a replacement gene encodingall or a portion of a variable region having a desired antigenspecificity and a target sequence which directs gene modification of allor a portion of the variable coding region in the host cell chromosome.After transfection, homologous recombination within the lymphoid cellline will occur; some of these recombination events will lead to thereplacement of all or a portion of the variable region of theimmunoglobulin gene with the replacement gene and, therefore, theexpression of chimeric antibody molecules by the transfected cells.

Once the transfectant that expresses the chimeric antibody isidentified, the practice of the invention involves culturing thetransfectant and isolating the chimeric antibody molecules from the cellculture supernatant using techniques well known in the art for isolatingmonoclonal antibodies. Alternatively, the transfected cells may becultured in ascites fluid in animals and harvested using well knowntechniques for isolating monoclonal antibodies.

In an alternate embodiment of the invention, homologous recombination invivo may be used to generate chimeric T cell receptor molecules.According to this embodiment, the replacement gene is targeted torecombine with a T cell receptor gene via a target gene sequencehomologous to a second DNA sequence adjacent to the T cell receptor genesequence to be modified.

The various aspects of the invention are described in more detail in thesubsections below and demonstrated by way of examples in whichmouse/human chimeric immunoglobulin heavy chains are produced. Forpurposes of clarity in discussion the invention will be described asfollows: (a) the target vectors; (b) transfection; and (c) screening andselection of transfectants which produce chimeric antibody molecules.

5.1. Target Vectors

As explained previously, the target vectors of the invention compriserecombinant DNA vectors including, but not limited to, plasmids, phages,phagemids, cosmids, viruses and the like which contain the replacementgene and the target sequence. As described in more detail below, thereplacement gene may comprise any of a number of genes that encode adesired structural product whereas the target sequence may varydepending upon the type of antibody molecule being converted and theanimal cell-type being transfected. The target sequence and thereplacement gene are positioned in the target vector so thattransfection of the appropriate antibody-producing cell line with thetarget vector results in targeted homologous recombination and sitespecific insertion of the replacement gene into the antibody gene.

The target vectors of the invention may contain additional genes whichencode selectable markers including but not limited to enzymes whichconfer drug resistance to assist in the screening and selection oftransfectants; alternatively the vectors of the invention may becotransfected with such markers. Other sequences which may enhance theoccurrence of recombinational events may be included as well. Such genesmay include but are not limited to either eucaryotic or procaryoticrecombination enzymes such as REC A, topoisomerase, REC 1 or other DNAsequences which enhance recombination such as CHI. Furthermore,sequences which enhance transcription of chimeric genes produced byhomologous recombination may also be included in the vectors of theinvention; such sequences include, but are not limited to, inducibleelements such as the metallothionine promoter (Brinster et al., 1982,Nature 296:39-42). Various proteins, such as those encoded by theaforementioned genes may also be transfected in order to increaserecombination frequencies. Various target sequences, replacement genes,and selectable markers which may be used in accordance with the methodof the invention are described below.

In preferred embodiments of the invention, the target vector comprisesrestriction enzyme cleavage sites such that non-mammalian sequences maybe excised from the target vector by cleavage with one or morerestriction enzymes which do not cleave the mammalian sequences of thevector. This permits the separation of mammalian sequences fromnon-mammalian sequences using standard techniques (including but notlimited to polyacrylamide gel electrophoresis followed by elution ofpolynucleotide fragments); the mammalian sequences may then be used fortransfection of lymphoid cells. The isolation of mammalian sequences isparticularly useful when heavy chain and light chain target vectors arecotransfected or sequentially transfected, as it eliminates thepossibility of homologous recombination occurring between non-mammaliantransfected sequences to produce aberrant gene constructions. Inpreferred specific embodiments of the invention, the heavy chaindissociation vector hγ₁ HC-D (FIG. 7) or the light chain dissociationvector LC-D (FIG. 8) may be used.

5.1.1. Target Sequence

The composition of the target sequence may vary depending upon whetherthe target plasmid is to be used to replace all or a portion of eitherthe variable or constant region genes of light chains or heavy chainsand, further, upon the animal species of the host cell to betransfected. More specifically, target sequences should be homologous tosequences which are adjacent to or which flank the coding region for theconstant or variable region, or the portion thereof, to be replaced oraltered.

For example, in a chromosome, mature heavy chain genes are comprised, attheir 5' termini, of the VDJ regions; i.e., the variable region (V),diversity region (D), and joining region (J) followed by any remaining Jregions which are not expressed (the number of J regions varies with thespecies), and intronic sequences. The central and 3' portion of the geneconsists of the constant region exons (flanked and interspersed withintronic and untranslated sequences) which may be one of various classes(e.g., mu, delta, gamma, epsilon, alpha) each of which is associatedwith its own adjacent switch region. Thus, the target sequence used totarget homologous recombination in the heavy chain gene of an antibodyproducing host cell may comprise a region that is homologous to anyportion of the antibody gene, depending on the desired alteration. Forexample, the target sequence for directing replacement of heavy chainconstant regions may comprise sequences homologous to sequences spanningany region up to and including or excluding the switch region commencingwith V, D or J and would be positioned accordingly in the constructionof the target vector; e.g., at a location 5' to the coding region of thereplacement gene. The actual target sequence that could be used may varydepending upon the animal species of the target host cell and the classof antibody expressed by the target host cell.

By contrast to the arrangement of heavy chain genes in a chromosome, themature light chain genes are composed of a VJ region at their 5'termini, intronic sequences, and a single constant region exon. Thus,the target sequence used to target homologous recombination in the lightchain gene of an antibody producing host cell may comprise a region thatis homologous to any portion of the gene, depending on the desiredalteration. For example, the target sequence for directing thereplacement of light chain constant regions may be homologous tosequences spanning all or portions of the appropriate V and J throughintronic sequences preceding the coding region for the constant regionof the light chain. Such target sequences would be appropriatelypositioned in the target plasmid; e.g., at a location 5' to the codingsequence for the replacement gene. Once again, the actual nucleotidesequence of the target sequence may vary with respect to the animalspecies of the target host antibody producing cell.

In addition to the sequences described above, the target sequence mayinclude sequences homologous to regions adjacent to the 5' and/or 3'terminus of the coding region for the constant heavy or light chain, andtherefore, would be positioned accordingly in the construction of thetarget vector; i.e., at a location 5' and/or 3', respectively, to thecoding region of the replacement gene. In a similar fashion, targetsequences for directing the replacement of heavy or light chain variableregions may comprise sequences homologous to all or portions of theappropriate regions that flank the variable region. In any case, targetsequences may also include coding region sequences flanking an areawithin an exon where only a portion of the variable or constant regionis to be replaced so that the protein expressed is altered in a desiredfashion.

5.1.2. Replacement Gene

As previously explained, the replacement genes used to convert antibodyconstant regions may comprise the coding sequence for all or a portionof a constant region of an immunoglobulin of a different class and/oranimal or human species. Thus, in the case of heavy chains, thereplacement gene may comprise all or a portion of the gene encoding theconstant regions of human IgM, IgD, IgG, IgE, and IgA, or any subclassthereof. Alternatively, the replacement gene may encode a product whichcan confer some different effector function to the chimeric moleculewhich would be expressed. For example, an enzyme, toxin, growth factor,biologically active peptide, linker, etc. The replacement gene may alsoconsist of any combination of aforementioned sequences, for example, allor a portion of an antibody constant region linked to a novel proteinsequence.

The replacement gene chosen depends, in part, upon the use intended forthe chimeric antibody molecule expressed. For example, if therapeuticuse in humans is intended, then the replacement gene could encode ahuman constant region, preferably of a class having a desired effectorfunction for the therapeutic use in mind. If an improvement oralteration in the existing effector function of the antibody is desired,a portion of the constant region may be replaced with a sequence thatconfers such improved or altered effector function to the resultingchimeric antibody molecule. If targeted delivery of an enzyme, toxin,drug, hormone or growth factor in vivo is desired, a replacement geneencoding the enzyme, toxin, drug, hormone or growth factor or anappropriate linker for conjugation to such should be used. If thechimeric antibodies are to be used in diagnostic assays, for examplewhere labeled antibodies are utilized, a replacement gene encoding anenzyme or its substrate could be used. Such enzyme/substrate systemsinclude, but are not limited to those which produce a colored productwhen reacted; for example, betagalactosidase, alkaline phosphatase,horseradish peroxidase, and the like. The resulting chimeric antibodiesmay be used as labeled antibodies in the procedures intended with orwithout further modification, e.g., the chemical attachment of enzymes,drugs, toxins, hormones, growth factors etc.

The replacement gene used to convert antibody variable regions maycomprise all or a portion of the coding sequence for a variable regionof an antibody molecule that defines a desired antigen. These may encodeantigen binding regions that define related or completely unrelatedantigens. If an improvement or alteration in antigen binding orspecificity, is desired, a portion of the variable region may bereplaced with a sequence that confers such improved or altered bindingor specificity to the resulting chimeric antibody molecule. Importantly,the replacement gene is not expressed unless it is integrated into thegenomic DNA of a host cell, thereby acquiring the necessary sequencesfor transcription or translation.

5.1.3. Selectable Marker and Other Elements

In addition to the target sequence and the replacement gene, the targetvector of the invention may encode a selectable marker which assists inthe screening and selection of antibody producing cells that have beensuccessfully transformed. Such markers include drug resistance genes,such as gpt, neo, his, DHFR etc., and the like.

Additional elements which may enhance the number of recombinationalevents may be included in the target vector. For example, an origin ofreplication (ori) that is temperature sensitive (for example polyoma tsAori system) may be included in the construct so that growth oftransfectants at a permissive temperature results in vector replicationso that the copy number of target sequence and replacement gene isincreased, and the subsequent number of recombinations may be increased.Other ori systems could also be utilized to increase copy number (e.g.,EBV ori plus factors, BPV ori plus factors, or SV40 ori and T antigen).

5.2. Transfection of Antibody-producing Cell Lines With Target Vectors

In accordance with the method of the invention, a cell line whichproduces a desired antibody (i.e., one having a desired antigenspecificity or a desired constant region) is transfected with theappropriate target vector to produce transfectants that will undergosite directed homologous recombination. Both light chain and heavy chaintarget vectors can be used to transfect an appropriate antibodyproducing cell line; however, in many cases transfection with a heavychain target vector may be sufficient to obtain expression of a chimericantibody molecule.

For example, lymphoid cell mutants, which comprise immunoglobulin genesencoding molecules of the desired antigen specificity or effectorfunctions, may express only immunoglobulin light chains or,alternatively, only immunoglobulin heavy chains. Lymphoid cell lineswhich express only light chains may be capable of producing antibody ofthe desired antigen specificity or effector function after transfectionof a vector which supplies heavy chain components; whether or not vectorencoding light chain components need be cotransfected may vary amongantibody/antigen systems. In Section 6, infra, transfection of the heavychain mutant mouse myeloma cell line J558L (which expresses only lightchains) with vectors supplying human and murine heavy chain, but notlight chain, immunoglobulin components resulted in transfectants capableof producing chimeric antibodies bearing human gamma 1 heavy chain.Section 9' infra, describes experiments in which a target plasmidcomprising human heavy chain sequence was transfected into murinehybridoma cell line L6' which produces monoclonal antibody directedtoward a tumor specific antigen. A transfected cell line was found tostably produce chimeric heavy chain in the absence of murine heavychain; the antibody produced by this cell line was found to bind tohuman tumor cells and to mediate destruction via antibody dependent cellmediated cytotoxicity with human effector cells more efficiently thanmurine L6.

Alternatively, it may be preferable to "chimerize" both light and heavyimmunoglobulin chains of a lymphoid cell line by cotransfecting thelymphoid cell line with target vector comprising heavy chain sequencesand target vector comprising light chain sequences. In preferredembodiments of the invention, when target vectors are cotransfected,each vector is designed so as to preclude homologous recombination fromoccurring with its covector (refer to Section 10, infra).

Transfection may be accomplished by any of a number of methods known tothose skilled in the art, including, but not limited to calciumphosphate precipitation, electroporation, microinjection, liposomefusion, RBC ghost fusion, protoplast fusion, etc. The target vector maybe linearized by cleavage with a restriction enzyme within the targetsequence prior to transfection in order to increase the probability ofhomologous recombination in the transfected cell.

5.3. Screening and Selection of Recombinants

The ultimate test for successful transformation, homologousrecombination and targeted gene modification is the production ofchimeric antibodies by the lymphoid cell line. The detection oftransfectants which produce chimeric antibodies can be accomplished in anumber of ways, depending upon the nature of the replacement geneproduct.

If the target vector contains a selectable marker, the initial screeningof transfected cells should be to select those which express the marker.For example, when using a drug resistance gene, those transfectantswhich grow in the selection media containing the otherwise lethal drugcan be identified in the initial screening. A second screening wouldthen be required to identify those transfectants which express thechimeric antibody.

The protocol for the second screening depends upon the nature of thereplacement gene. For example, the expression of a replacement gene thatencodes the constant region of a different antibody class or species canbe detected by an immunoassay using antibodies specific for theparticular immunoglobulin class and/or species; alternatively, abioassay could be performed to test for a particular effector functionconferred by the replacement gene. The expression of a replacement genewhich encodes a biologically active molecule such as an enzyme, toxin,growth factor, or other peptide could be assayed for the particularbiological activity; for example, the transfected cell products can betested using the appropriate enzyme substrate, or target for the toxin,growth factor, hormone, etc.; alternatively, these replacement geneproducts could also be assayed immunologically using antibodies whichare specific for the replacement gene product.

According to a preferred specific embodiment of the invention, thefollowing selection procedure may, for example, be employed. Lymphoidcells may be transfected with target vector comprising human constantregion sequences, as described supra, and then cultured in a singleflask of selective media. After a period of time sufficient to allowselection to occur (in most cases, about 2 weeks) the surviving cellsmay then be treated with antibody that is specific for murineimmunoglobulin constant region (and preabsorbed against humanimmunoglobulin) and is conjugated to a toxin, such as ricin. Cells whichhad not been successfully transfected would continue to express antibodycontaining murine constant region sequences, and would be killed by thetoxin conjugated to the anti-murine immunoglobulin antibody. Cellsexpressing chimeric antibodies which contain human constant regionsequences would survive, and could subsequently be cultured in soft agarand be further identified by an overlay of anti-human immunoglobulinantibody.

The transfectants which express a replacement gene should also be testedfor appropriate antigen recognition. This can be accomplished by anappropriate immunoassay, including a competitive immunoassay using theoriginal and chimeric antibodies. These screening tests need not becarried out sequentially and, in fact, could be conducted simultaneouslyusing a "sandwich immunoassay" in which a capture antibody that definesthe replacement gene product (i.e., either the constant or variableregion) is used to immobilize the chimeric antibody and the presence orabsence of the unaltered portion (i.e., either the unaltered variableregion or unaltered constant region, respectively) is detected (i.e.,using labeled antigen or labeled antibody, respectively). For example,the antigen could be used to capture the chimeric antibodies and theconstant region replacement gene product could be detected using labeledantibodies that define the replacement gene product, or by assaying thecaptured chimeric antibodies for the biological activity of thereplacement gene product (e.g., enzymatic, toxin, hormone, growthfactor, etc.). Alternatively, the chimeric antibody can be immobilizedat the constant region (e.g., using an antibody specific for thatregion, or staphylococcal A protein, etc.) and the variable region geneproduct could be detected using labeled antigen or an anti-idiotypeantibody.

If the lymphoid cell line is incapable of producing functional antibody(for example, because it is capable only of producing light chain),following transfection with the target vectors of the invention, usefultransfectants may be identified by assays which identify functionalantibody, including, but not limited to, CDC, ADCC, orimmunoprecipitation reactions.

6. EXAMPLE: REPLACEMENT OF THE CONSTANT REGION OF A MURINEIMMUNOGLOBULIN HEAVY CHAIN WITH THE CONSTANT REGION OF HUMAN GAMMA 1IMMUNOGLOBULIN

The examples that follow describe the construction of a target plasmidcontaining a murine target sequence (encoding the fourth joining regionand enhancer of the heavy chain gene) ligated to a replacement geneencoding the constant region of a human gamma 1 immunoglobulin (huIgG1).This target plasmid was used along with a phage containing the entiremature gene encoding a murine heavy chain (the entire variable andconstant region) to co-transfect murine myeloma cells which are heavychain mutants that ordinarily express only light chains. The transfectedcells were screened and clones expressing the human IgG1 heavy chainwere identified. These experiments indicate the successful homologousrecombination event, integration into the host cell chromosomes, andexpression of the human gamma 1 gene in a murine cell system.

6.1. Materials and Methods

Unless otherwise indicated, the materials and methods indicated belowwere used in the examples that follow.

6.1.1. Transfection

The transfections were carried out by washing and resuspending about 10⁷cells in PBS (phosphate buffered saline) at 4° C. with 2 mM MgCl₂ and 2mM CaCl₂, and transferring the cells to a sterile plastic cuvette linedwith aluminum foil (BioRad, Calif.). The linearized DNA was added andmixed at a final concentration of 10-100 ug/ml, then an electricalcharge was applied with an appropriate power supply (e.g., a GenePulsar, BioRad, Calif.). The cuvettes were gently "flicked" to insuregood mixing. After 2 minutes at room temperature the cells were thentransferred to 9 ml of RPMI media (GIBCO) with 10% FBS at 37° C. After48 hours incubation, viable cells were recovered by centrifugation,counted, and plated out at a density of 10³ cells/well in 96 well platesor at 10⁴ cells per well in 24 well plates in RPMI media with 10% FBS,penicillin (60 mg/ml)/ streptomycin (100 mg/ml), sodium pyruvate (1 mM),L-glutamine (292 mg/ml), and 0.1-2 ug/ml mycophenolic acid or 1-2 mg/mlG418. The same media was either replenished or exchanged every 2 to 3days. After 2 to 3 weeks, wells were scored for growth. Supernatantsfrom cultures were then assayed by ELISA for the presence of human gamma1 and positive wells were cloned and screened again.

6.1.2. Screening Transfectants for Successful Recombinants

ELISA assays were performed by coating immulon 2 plates (Dynatec Labs,Chantilly, Va.) with 100 ul of either goat anti-human IgG (AntibodiesInc., Davis, Calif.) at a 1:1000 dilution, or goat anti-mouse IgA(Cappel) at a 1:5000 dilution in coating buffer (0.1 M NaHCO₃ pH 9.6) at4° C. overnight. Plates were then filled with specimen diluent (GeneticSystems) to block for one hour at room temperature after which they wereshaken out, sealed, and stored at 4° for no more than 3 weeks. Theplates were readied for assay by washing 3 times with wash buffer (0.15M NaCl, 0.05% v/v Tween 20) and then 100 ul of standards (diluted in theappropriate media) or culture supernatants were added and incubated at37° C. for 1 hour. The plates were then washed 3 times with wash bufferand bound antibody was detected with 100 ul of either horseradishperoxidase (HRP) conjugated goat anti-human IgG (American Qualex) at a1:6000 dilution, HRP goat anti-mouse IgA (Cappel) at a 1:5000 dilution,or HRP goat anti-mouse Lambda (Southern Biotech. Ass. Inc., Birmingham,Ala.) at a 1:3000 dilution, for 1 hour at 37° C. Plates were then washed3 times with wash buffer and incubated at room temperature for 15minutes with a 1:100 dilution of TMB Chromogen in buffered substrate(Genetic Systems), stopped with 100 ul per well of 3M H₂ SO₄, and readimmediately at 450/630 nm on a micro plate reader (Genetic Systems).Likewise, Kappa producing cell lines can be screened using an HRP goatanti-mouse Kappa reagent (Southern Biotech. Ass. Inc.). Cell linespositive for human IgG and/or murine IgA were subcloned by limitingdilution or in soft agarose to isolate expressing clones. To clone insoft agarose, approximately 1000 cells from a positive well wereresuspended in 0.4% agarose and layered over an agar layer of murineperitoneal exudate feeder cells. A third layer containing antiserumspecific for human IgG1 was overlayed 1 to 2 days later. Positive cloneswere identified by the presence of an immunoprecipitate.

6.1.3. Southern and Western Blots

High molecular weight DNA was isolated from cells essentially asoriginally described by Blin and Stafford (Blin. N. and Stafford, D. W.,1976, Nucleic Acids Res. 3:2303) and later by Maniatis, Fritsch andSambrook (Maniatis et al., 1982, Molecular Cloning--A Laboratory Manual,Cold Spring Harbor, N.Y. p. 280) with the exception that the cells werefirst washed in standard phosphate buffered saline (as opposed to Tris)and the RNase treatment was carried out simultaneously with theproteinase K treatment at 55° C.

Southern blots and hybridization were performed as originally describedby Southern (Southern, 1975, J. Mol. Biol. 98:503) and more recentlydetailed by Maniatis, Fritsch, and Sambrook (Maniatis et al., 1982,Molecular Cloning--A Laboratory Manual, Cold Spring Harbor, N.Y., pp.383-389). Probes used for hybridization were the 1.0 kb Hind III-PstIfragment containing the first constant region domain of the human IgG1gene, or the 0.7 kb Hind III-Hind III fragment encoding the murineJ_(H2) and J_(H3) gene segments. Labeling of probes was performed as perthe manufacturers protocol using a Nick Translation Kit (BethesdaResearch Laboratories). Restriction enzymes were purchased fromBoehringer Mannheim Biochemicals.

Western blot analysis was performed as described (Towbin et al., 1979,Proc. Natl. Acad. Sci. (USA) 76:4350) and the developing reagents arethe same as those used for detection in the ELISA assay.

6.2. Construction of Target Plasmids Encoding Human ImmunoglobulinConstant Region

A target plasmid was constructed which consists of the bacterial plasmidvector pRSV-140-neo into which a 8.0 kb Hind III- Bam HI fragmentcontaining the human gamma 1 constant region gene was cloned as thereplacement gene (FIG. 2). The target gene, a 2.2 kb Hind III fragmentderived from the murine immunoglobulin heavy chain (IgH) locus whichcontains the fourth joining region (J4), the IgH enhancer (e), andintronic sequences 5' to the switch region, was inserted into the HindIII site located 5' to the human gamma 1 gene (FIG. 2). This constructwas then linearized at the unique Xba I site within the murine targetsequence and transfected into the murine myeloma cell line describedbelow by means of electroporation previously described.

6.3. Transfection and Homologous Recombination

The experiments and results described below demonstrate successfulhomologous recombination between the target plasmid and the murine heavychain gene contained within the phage which was used to co-transfect amurine myeloma host cell. Homologous recombination and integration intothe murine myeloma host cell chromosome resulted in the expression of ahuman immunoglobulin heavy chain (IgG1).

6.3.1. Co-transfection of Murine Myeloma Cells

The target plasmid containing (a) the murine target sequence encodingthe fourth joining region (J4) the IgH enhancer, and intronic sequences5' to the switch region, and (b) the replacement gene encoding the humanIgG1 constant region, was linearized in the region of the targetsequence and co-transfected at an equimolar ratio with a phage DNA clonecontaining the functional J558 murine heavy chain gene, into the heavychain mutant mouse myeloma cell line, J558L which expresses only lightchains. Transfected cell lines resistant to G418 (the selectable markerencoded by the target plasmid construct) were then screened for thepresence of human IgG1 protein. Since the human gamma 1 constant regioncontained in the target plasmids constructed above lacks theimmunoglobulin promoter sequence, the detection of expression of humanIgG protein by the transfected mouse cell line should be the result ofan homologous recombination event between the transfected DNA moleculesand integration within the host cell chromosome.

6.3.2. Identification of Cells Secreting Chimeric Immunoglobulins

Transfectants secreting human IgG and/or murine IgA protein weresubcloned. Confirmation of the homologous recombination events wasprovided by DNA blot transfer analysis, in which a newly rearrangedrestriction fragment was identified; this fragment was of the sizeexpected (a 10.5 Kb Bam HI fragment) and contained both human gamma 1,and mouse J_(H2) -J_(H3) sequences. Additional confirmation was obtainedby Western blot analysis of supernatant proteins which demonstrated aprotein chain of about 50 kilodaltons (50 kd) bearing human IgGserologic determinants present in the transfectoma culture supernatants.The antihuman IgG reagent did not react with murine IgA, which was alsodistinguishable based on slower electrophoretic mobility.

ELISA results also revealed the presence of murine IgA in manytransfectoma supernatants. This is the result of functional J558 heavychain gene integrating undisrupted (i.e., without undergoing anhomologous recombination event). By comparing the level of human IgG andmouse IgA expression and production, the frequency of homologous versusnonhomologous recombination events has been estimated to be between30-80%, depending on the experiment.

The results presented above have conclusively demonstrated: (a) that thetarget plasmid works as designed; and (b) that myeloma cells are veryefficient in their ability to mediate homologous recombination.

Sequences homologous to regions upstream and/or downstream of variableregion gene segments could be used in conjunction with variable regiongene segments or portions thereof to alter the antigen affinity and/orspecificity. Moreover, the J558 system described herein can be used as ascreening procedure for identifying recombination enhancing proteins.

7 EXAMPLE: PRODUCTION OF CHIMERIC IMMUNOGLOBULIN HEAVY CHAIN WITHSPECIFICITY TO L20 HUMAN TUMOR ASSOCIATED ANTIGEN

The experiments described below demonstrate that murine hybridoma cellscan efficiently mediate homologous recombination and that thiscapability can be exploited to direct major reconstructions of theendogenous immunoglobulin heavy chain locus. A plasmid was constructedcontaining the human IgG1 constant region exons flanked by the murineheavy chain enhancer and the neomycin gene. This construct was used todirect the production of antigen specific chimeric heavy chain Ig bysite specific targeting to the endogenous heavy chain locus of thehybridoma cell line L20 (deposited with the American Type CultureCollection (ATCC) and having Accession Number HB8913) which produces amonoclonal antibody specific for a human tumor associated antigen(Patent Application No. 834,172, filed Feb. 26, 1986 and referencesincorporated therein). The frequency of the targeting event is observedto be 1 in 200 or 0.5% of the hybridoma cells which integrate theplasmid.

7.1. Construction of Target Vector and Transfection of Hybridoma L20

A plasmid vector was constructed which contains the constant regionexons of human IgG1 (Cg1) flanked by the murine heavy chain enhancer(MHE), and the neomycin resistance gene (NEO) (FIG. 3). The plasmid waslinearized at a unique Xba I site within the 2.2 kb region of sequenceidentity shared with the murine IgH locus, and transfected into thehybridoma cell line L20 which produces a murine IgG1 antibody specificfor an antigen expressed primarily on human carcinoma cells as follows:The vector described in FIG. 3 was linearized at the unique Xba I siteand 50 ug was used to transfect 8×10⁶ L20 hybridoma cells byelectroporation. Viable cells were plated at a density of 1×10⁴ (or1×10³ for frequency calculation) cells/well in 96 well plates in IMDM10% FBS media containing 2.5 mg/ml G418. The plates were fed every 2-3days and after 2 weeks all wells were screened for the production ofhuman IgG1 using standard ELISA techniques. Goat anti-human IgG (Cat.#4132 Antibodies Inc., Davis Calif.) at a 1:10,000 dilution was used asa capture reagent, and goat anti-human IgG/HRP (Cat #HAP009 AmericanQualex, La Mirada Calif.) at a 1:6000 dilution was used to detect. Allpositive supernatants were verified in subsequent ELISAs as well.

Since the human IgG1 exons are not associated with a variable regiongene segment, production of human IgG1 heavy chain protein can onlyoccur by recombination of the plasmid with a functional promoter,initiation codon, and splicing sequences. Thus, cells survivingselection in G418 containing media (i.e., all those which satisfactorilyintegrated the plasmid) were assayed for production of human IgG1 byELISA (Table I).

                  TABLE I                                                         ______________________________________                                        FREQUENCY OF INTEGRATION EVENTS                                               WHICH RESULT IN HUMAN IgGl PRODUCTION                                         INT                                                                           EVENTS   WELLS      TOTAL     WELLS W/                                        PER WELL SCREENED   EVENTS    HU IgGl  FREQ                                   ______________________________________                                        2.2      480        1056      8        0.75%                                  ______________________________________                                    

The frequency of transfection was determined by plating at lowerdensity, and the frequency of integration events resulting in productionof human IgG1 was therefore observed to be on the order of 1 in 132 or0.75%. More recent experiments with the same cells and plasmid resultedin the production of human IgG in 2 of 512 integrations, or 0.39%--thesesuggest that an average frequency may be closer to 0.5%. Interestingly,even after cloning, most cell lines were found to produce both murineand human IgG1, although occasional clones were isolated which producedone or the other (Table II). Cells from parent wells producing humanIgG1 were plated at limiting dilution and 12 clones from each parentwere assayed for production of human IgG1 (huIgG1) (as described forTable 1) and mouse IgG. Mouse IgG (mIgG) assays were carried out usinggoat anti-mouse IgG (Cat #1070 Southern Biotech, Birmingham Ala.) andgoat anti-mouse Kappa/HRP (Cat #OB1141-85 Fisher Biotech, OrangeburgN.Y.) as capture and detecting reagents respectively. Results are shownin Table II. Numbers given are of clones producing human antibody,mouse, or both.

                  TABLE II                                                        ______________________________________                                        PRODUCTION OF MOUSE vs HUMAN                                                  IgGl IN CLONES FROM L20                                                       TRANSFECTANTS PRODUCTING HUMAN IgGl                                           PARENT    huIgGl      mIgG    huIgGl/mIgG                                     ______________________________________                                        7C5       0           0       12                                              7E6       1           0       11                                              8D6       0           0       12                                              8F8       2           2        8                                              8H12      1           0       11                                              10G3      0           2       10                                              9F11      0           1       11                                              9F12      1           0       11                                              ______________________________________                                    

7.2. Characterization of Expressed Chimeric L20 Heavy Chain 7.2.1.Western Blot Analysis

The human heavy chain was shown to be serologically distinct and in theexpected size range by Western blot analysis of supernatants fromselected clones as follows: cultures nearing the plateau portion ofgrowth were washed and resuspended in serum free media. After 24 hoursthe supernatants were harvested, dialyzed vs. 0.1 M NH₄ OAc,lyophilized, and resuspended in 5 mM Tris, pH 6.8. Aliquots of thesesamples and the parent cell line L20, plus purified antibody 2H9 (ahuman IgG1 protein), and purified murine L20 IgG1 were denatured byboiling in the presence of β-mercaptoethanol and electrophoresed througha gradient acrylamide gel ranging between 10 to 20%. The gel wastransferred to nitrocellulose paper electrophoretically. The resultingfilter was blocked with 2% nonfat dry milk in PBS, stained with goatanti-human IgG/HRP (#10501 CALTAG, San Francisco Calif.) and developedwith 30 mg 4-chloro-1 napthol (Sigma, St. Louis Mo.) in Tris bufferedsaline.

7.2.2. Flow Cytometric Analysis

Both the human and murine IgG1 were also demonstrated to be antigenspecific by flow cytometry (Table III) as follows: The samples used forwestern blotting were assayed by ELISA to determine the concentration ofmurine and human IgG1. Dilutions were made to adjust the concentrations(in ug/ml) to those given for flow cytometry analysis. Five×10⁵ cellsfrom the human tumor lines 2981 or 3347 (Hellstrom et al., 1986, Proc.Natl. Acad. Sci. USA 83:7059-7063) were incubated in the presence of theL20 antibodies indicated (or media) for 30 minutes at 4° C., washed 2×,and stained with a 1:50 dilution (in media) of either goat anti-humanIgG/FITC (fluorescein isothiocyanate) (CALTAG, San Francisco Calif.) orgoat anti-mouse IgG/FITC (TAGO, Burlingame Calif.--heavy and light chainspecific) for 30 minutes at 4° C. These preparations were then washedtwice, resuspended, and analyzed by flow cytometry for relativefluorescence. Results are shown in Table III. The values given are thelinear fluorescence equivalence (LFE) of each preparation.

                  TABLE III                                                       ______________________________________                                        ANTIGEN SPECIFICITY OF MURINE AND                                             HEAVY CHAIN CHIMERIC L20 ANTIBODIES                                                    Ab conc       LFE                                                    CLONE      huIgG   mIgG        α-hu                                                                         α-m                                 ______________________________________                                                                       2981 Target                                    7E6-10     1.0     0           35   6                                         8H12-8     1.0     0           38   7                                         8F8-8      1.0     0.5         39   11                                        9H12-1     1.0     0.1         42   7                                         8F8-10     0       0.4         2    10                                        10G3-5     0       1.0         2    31                                        mL20       0       1.0         2    18                                        media      0       0           2    2                                                                        3347 Target                                    7E6-10     0.4     0           7    3                                         mL20       0       0.4         2    3                                         media      0       0           2    2                                         ______________________________________                                    

Thus, the parental L20 cell line most probably retains two copies of theproductive heavy chain allele, only one of which undergoes a specificrecombination within a given cell. The production levels for clonesexpressing only the chimeric antibody was found to be on the order of5-10 ug/ml in 10 ml culture supernatants at the plateau phase of growth.

7.2.3. Southern Blot Analysis

Southern blot analysis using a probe specific for human Cγ1 demonstratedintegration specific fragments in common between all cell linesproducing human IgG1, with three different enzymes. A Bam HI site wasmapped 1.4 kb upstream of the target sequence in the L20 parental line,therefore an insertion of the recombination vector at the target site isexpected to produce a Bam HI fragment of 5.5 kb which hybridizes to ahuCγ1 probe. Such a fragment was observed in each clone producing humanIgG1.

7.2.4. ADCC Analysis

The murine L20 IgG1 antibody was compared with the protein bearing thechimeric heavy chain for activity in an ADCC assay (Hellstrom et al.,1985, Proc. Natl. Acad. Sci. USA 82:1499-1502). As can be seen from theresults in Table IV, changing the heavy chain constant regions to thoseof human IgG1 provides a novel effector function to this specificity.

                  TABLE IV                                                        ______________________________________                                        ADCC OF 2981 TARGET CELLS MEDIATED BY                                         MURINE MAb L20 AND CHIMERIC HEAVY CHAIN L20                                                  Conc     ADCC of                                               Immunoglobulin (μg/ml)                                                                             2981 Target Cells                                     ______________________________________                                        L20 8F8-8      4        58%                                                   L20 7E6-10     8        53%                                                   MAb L20        10       31%                                                   None*          0        31%                                                   ______________________________________                                         *Effector cells (lymphocytes) only.                                      

7.3. Chimeric L20 Heavy Chain Produced Using a Different Target Vector

In further examples we have used another plasmid, which has a 2.8 kbfragment encoding a human c gamma 1 (as described in FIG. 4) tointroduce the human Cγ1 exons into murine hybridoma cells L20 resultingin the production of human IgG1 chimeric heavy chain proteins. Thisplasmid was transfected into hybridoma cell line L20 resulting in 4wells producing huIgG in an ELISA assay of approximately 700 integrationevents.

8. CONSTRUCTION OF CHIMERIC G28.1 HEAVY CHAINS

In another example we have used the plasmid shown in FIG. 3 to convertthe murine hybridoma cell line G28.1 (Ledbetter et al., Leukocyte typingIII, A. J. McMichael, ed., Oxford Univ. Press pp.339-340 (1987)) to theproduction of antigen specific human IgG1 heavy chain by the samemethod. A single experiment resulted in 4 huIgG1 producers out of 864integration events. These cell lines were identified by ELISA, verified,and then tested for their ability to bind to tonsilar B cells asfollows: human tonsilar B cells were incubated with supernatant from thecell lines indicated (or Media), washed and counterstained with eithergoat anti-human IgG (α hu) or goat anti-mouse IgG (α m) as previouslydescribed, and analyzed by flow cytometry. Results are shown in Table V.The supernatants were quantitated by ELISA for mouse and human IgG andvalues are given in ng/ml. Fluorescence data are expressed as linearfluorescence equivalents.

                  TABLE V                                                         ______________________________________                                        ANTIGEN SPECIFICITY OF HEAVY                                                  CHAIN CHIMERIC G28.1 SUPERNATANTS                                             CELL LINE   huIgG       mIgG    LFE                                           ______________________________________                                                                        α-hu                                    11F10        0          87       9                                            1F11        97          89      93                                            4F5         86          91      38                                            3D2         90          86      77                                            3D11        56          85      16                                            Media        0           0       9                                                                            α-m                                     G28.1        0          83      53                                            Media        0           0       3                                            ______________________________________                                    

The results again demonstrate preservation of antigen specificity for anantibody molecule bearing a human IgG1 chimeric heavy chain.

9. CONSTRUCTION OF CHIMERIC L6 HEAVY CHAINS

The recombinant target plasmid depicted in FIG. 4B was transfected intomurine hybridoma cell line L6, (deposited with the ATCC and havingAccession No. HB8677) resulting in a single cell line which give rise toclones stably producing chimeric heavy chain (in the absence of murineheavy chain). The antibody was shown to bind to human tumor cells and tomediate destruction via ADCC with human effector cells more efficientlythan murine L6 (Table VI), comparable to that of heavy and light chainchimeric L6 produced by conventional recombinant techniques (PatentApplication No. 684,759, filed Dec. 21, 1984 and Patent Application No.776,321, filed Oct. 18, 1985 and references incorporated therein).

                  TABLE VI                                                        ______________________________________                                        ADCC OF 2981 TARGET CELLS MEDIATED BY                                         MURINE MAb L6 AND CHIMERIC HEAVY CHAIN L6                                                    Conc.    ADCC of                                               Immunoglobulin (μg/ml)                                                                             3347 Target Cells                                     ______________________________________                                        L6 7B7.16      0.01     83%                                                   L6 7B7.7       0.01      82.5%                                                MAb L6         0.01     58%                                                   None*          0.01     53%                                                   ______________________________________                                         *Effector cells (lymphocytes) only.                                      

Southern blots confirmed the presence of a 10 kb Ava I fragment, and an8 kb Bgl II fragment, both of which hybridize to a huCg1 probe. Thesefragments are consistent with insertion of the vector plasmid at thetarget site based on previous mapping of the cloned genomic L6 heavychain variable region gene segment.

10. VECTORS USEFUL FOR PRODUCTION OF CHIMERIC ANTIBODIES

Complete "chimerization" of an antibody molecule requires replacement ofthe light chain constant region as well as the heavy chain. Therefore,vectors were generated to effect chimeric construction of murine kappalight chains by homologous recombination. The first such construct (FIG.5) was comprised of a 2.7 kb Eco RI fragment containing the exon forhuman Cκ, and, as the target sequence, the 2 kb Pst I/Xmn I fragmentcontaining the murine light chain enhancer from the murine light chainintron separating the variable and constant region exons. The vectoralso contained the bacterial guanosine phosphoryl transferase (gpt) genederived from the pSV₂ -gpt plasmid (for selection in eukaryotic cells)as well as the ampicillin resistance gene and the bacterial origin ofreplication from pUC18. A second version of the vector was generated byreplacing the gpt gene with a mutated version of the dihydrofolatereductase gene (*DHFR) (FIG. 6). This mutated form of DHFR wasoriginally described by Simonsen and Levinson (1983, Proc. Natl. Acad.Sci. U.S.A. 80:2495-2499) as having a lower affinity for the drugmethotrexate than does the wild type gene and which therefore can beused for selection in cells which retain the normal DHFR gene. Stepwiseincrease of methotrexate concentration has been used to effect thegenetic amplification of both DHFR and linked genes with concomitantincrease in production of protein encoded by the linked gene. Thus, themutated DHFR vector offers the advantage of boosting levels ofimmunoglobulin production via gene amplification.

Both the light chain vectors have been used to create chimeric lightchain in the BR64 hybridoma cell line. Transfections were performed asdescribed supra. Eleven cell lines were obtained from transfection withthe gpt vector and >25 with the DHFR vector.

However, transfection of the gpt vector into the HCCL20 cell line didnot yield any human kappa light chain producers in three transfectionexperiments. Bacterial vector sequences (i.e. amp and the bacterialorigin of replication) and SV40 regulator sequences flanking theselectable gene (neo and gpt) are shared by both the human gamma 1 heavychain recombination vector and the light chain vectors. These sequenceswhich are shared may serve as target sequences in sequentialtransfections, thus decreasing the frequency with which targeting occursat the murine light chain locus. Therefore, vectors were designed whichbore much less homology to each other and could be separated from thebacterial sequences necessary for production of the plasmid byrestriction enzyme digestion.

The human gamma 1 heavy chain dissociation vector (FIG. 7) consists ofthe 1.3 kb Xba I/Hind III fragment containing the murine heavy chainenhancer just upstream of the 2.9 kb Hind III/Pvu II fragment bearingthe human gamma 1 constant region exons. The neo gene was derived frompMC1 pol A vector (M. Cappechi) which has a synthetic polyadenylationsite following neo (Mansour et al., 1988, Nature 336:348-352), and wascloned just downstream of the gamma 1 exons as a 1.1 kb Xho I/Bam HIfragment. The remainder of the target sequence comprises the 0.9 kb HindIII/Xba fragment that contains the murine J_(H4) exon. This fragment waslocated just downstream of the neo gene followed by the bacterialsequences of pIC2OR. Upon digestion of this vector with the restrictionenzyme Xba I, the human gamma 1 exons become dissociated from thebacterial sequences, but retain the selectable neo gene and the targetsequences at either end in the same orientation as for the previousheavy chain vectors.

A BR64 human light chain producing cell line was chosen from theexperiments described above using the pSV₂ D^(R) /C.sub.κ vector (shownin FIG. 6) and transfected with the human heavy chain dissociationvector (hg₁ HC-D shown in FIG. 7). Twenty-eight cell lines were obtainedfrom this transfection that expressed both human gamma 1 heavy chain andhuman kappa light chain. Culture supernatants from several of these weretested for antigen specificity by incubating RCA tumor cells withculture supernatants, followed by washing and staining with goatanti-human IgG antiserum conjugated to fluoroscein. Many of thesupernatants that scored positive in ELISA for human IgG productionshowed binding to the human tumor cell line while supernatant from thehuman light chain producing parent cell was not recognized by antiseraspecific for human immunoglobulin, thus providing convincing evidence ofsuccessful chimerization with retention of antigen specificity. Twoclonal cell lines were generated that exhibited a production level ofchimeric antibody exceeding 70 μg/ml, an extremely high level ofproduction.

11. DEPOSIT OF MICROORGANISMS

The following plasmids were deposited with the Northern RegionalResearch Center (NRRL), and assigned the following accession numbers:

    ______________________________________                                        plasmid        host    accession number                                       ______________________________________                                        pCMV/huγ1                                                                              DH5α                                                                            B-18596                                                pSV.sub.2 gpt/C.sub.κ                                                                  DH5α                                                                            B-18597                                                pSV.sub.2 D.sup.R /C.sub.κ                                                             DH5α                                                                            B-18598                                                hγ.sub.1 HC-D                                                                          DH5α                                                                            B-18599                                                ______________________________________                                    

The present invention is not to be limited in scope by the embodimentsdisclosed in the examples which are intended as but single illustrationsof different aspects of the invention and any methods which arefunctionally equivalent are within the scope of this invention. Indeed,various modifications of the invention in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description. Such modifications are intended to fallwithin the scope of the appended claims.

What is claimed:
 1. A method for the production of a cell lineexpressing a chimeric antibody molecule, comprising:(a) transfecting anantibody-producing lymphoid cell line with a first and second targetvector, the first vector comprising:(i) a replacement gene to modify aportion of the genomic sequence of an immunoglobulin heavy chain gene ofthe lymphoid cell line, and (ii) a target sequence homologous to a DNAsequence adjacent to the immunoglobulin heavy chain sequence to bemodified, and the second vector comprising:(i) a replacement gene tomodify a portion of the genomic sequence of an immunoglobulin lightchain gene of the lymphoid cell line and (ii) a target sequencehomologous to a DNA sequence adjacent to the immunoglobulin light chainsequence to be modified, such that the first vector modifies the heavychain gene of the cell line by site specific homologous recombinationwith genomic chromosomal DNA in vivo and the second vector modifies thelight chain gene of the cell line by site specific homologousrecombination with genomic chromosomal DNA in vivo; and (b) selecting atransfectant which produces the chimeric antibody molecule.
 2. A methodfor the production of a chimeric antibody molecule, comprising:(a)culturing a chimeric-antibody-producing cell line which was prepared bytransfecting an antibody-producing lymphoid cell line with a first andsecond target vector, the first vector comprising:(i) a replacement geneto modify a portion of the genomic sequence of an immunoglobulin heavychain gene of the lymphoid cell line, and (ii) a target sequencehomologous to a DNA sequence adjacent to the immunoglobulin heavy chainsequence to be modified, and the second vector comprising:(i) areplacement gene to modify a portion of the genomic sequence of animmunoglobulin light chain gene of the lymphoid cell line and (ii) atarget sequence homologous to a DNA sequence adjacent to theimmunoglobulin light chain sequence to be modified, such that the firstvector modifies the heavy chain gene of the cell line by site specifichomologous recombination with genomic chromosomal DNA in vivo and thesecond vector modifies the light chain gene of the cell line by sitespecific homologous recombination with genomic chromosomal DNA in vivo,and the chimeric antibody is expressed by the transfected cell line; and(b) isolating the chimeric antibody molecule from the culture.